Polynucleotide probes for detection and quantitation of actinomycetes

ABSTRACT

Polynucleotide probes and accessory helper oligonucleotides useful for detecting the subset of High (G+C) subset of Gram-positive bacteria known as the &#34;Actinomycetes.&#34; The hybridization probes are highly specific for the Actinomycetes and do not cross-hybridize with the rRNA or rDNA of numerous other bacterial and fungal species.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/132,412, filed May 3, 1999. The disclosure of this relatedapplication is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to nucleic acid detection systems. Morespecifically, the invention relates to polynucleotide probes havingbinding specificity for rRNA or rDNA of the subset of Gram-positivebacteria known as the “Actinomycetes.”

BACKGROUND OF THE INVENTION

The Actinomycetes, or the “High (G+C)” subset of Gram-positive (Gram⁽⁺⁾)bacteria, are a distinct evolutionary lineage within the eubacteria. TheActinomycetes exhibit highly unusual phenotypic features as a reflectionof a characteristically high mutation rate. Many members of this groupof bacteria produce antibiotics and are commonly found in soil.Actinomycetes are responsible for a variety of significant animaldiseases including tuberculosis, leprosy, diphtheria and periodontaldiseases. Notably, immunodeficient individuals are particularlysusceptible to infection by the Mycobacteria avium, Mycobacteriaintracellulare, and Mycobacteria scrofulaceum, all of which areindividual species among the Actinomycetes. Additionally, theActinomycetes are responsible for a variety of economically importantplant diseases.

It is well established that two single strands of deoxyribonucleic acid(“DNA”) or ribonucleic acid (“RNA”) can associate or “hybridize” withone another to form a double-stranded structure having two strands heldtogether by hydrogen bonds between complementary base pairs. Theindividual strands of nucleic acid are formed from nucleotides thatcomprise the bases: adenine (A), cytosine (C), thymine (T), guanine (G),uracil (U) and inosine (I). In the double helical structure of nucleicacids, the base adenine hydrogen bonds with the base thymine or uracil,the base guanine hydrogen bonds with the base cytosine and the baseinosine hydrogen bonds with adenine, cytosine or uracil. At any pointalong the chain, therefore, one may find the classical “Watson-Crick”base pairs A:T or A:U, T:A or U:A, and G:C or C:G. However, one may alsofind A:G, G:U and other “wobble” or mismatched base pairs in addition tothe traditional (“canonical”) base pairs.

A double-stranded nucleic acid hybrid will result if a firstsingle-stranded polynucleotide is contacted underhybridization-promoting conditions with a second single-strandedpolynucleotide having a sufficient number of contiguous basescomplementary to the sequence of the first polynucleotide. DNA/DNA,RNA/DNA or RNA/RNA hybrids may be formed under appropriate conditions.

Generally, a probe is a single-stranded polynucleotide having somedegree of complementarity with the nucleic acid sequence that is to bedetected (“target sequence”). Probes commonly are labeled with adetectable moiety such as a radioisotope, an antigen or achemiluminescent moiety.

Descriptions of nucleic acid hybridization as a procedure for detectingparticular nucleic acid sequences are given by Kohne in U.S. Pat. No.4,851,330, and by Hogan et al., in U.S. Pat. Nos. 5,541,308 and5,681,698. These references also describe methods for determining thepresence of RNA-containing organisms in a sample which might containsuch organisms. These procedures require probes that are sufficientlycomplementary to the ribosomal RNA (rRNA) of one or more non-viralorganisms or groups of non-viral organisms. According to the method,nucleic acids from a sample to be tested and an appropriate probe arefirst mixed and then incubated under specified hybridization conditions.Conventionally, but not necessarily, the probe will be labeled with adetectable label. The resulting hybridization reaction is then assayedto detect and quantitate the amount of labeled probe that has formedduplex structures in order to detect the presence of rRNA contained inthe test sample.

With the exception of viruses, all prokaryotic organisms contain rRNAgenes encoding homologs of the procaryotic 5S, 16S and 23S rRNAmolecules. In eucaryotes, these rRNA molecules are the 5S rRNA, 5.8SrRNA, 18S rRNA and 28S rRNA which are substantially similar to theprokaryotic molecules. Probes for detecting specifically targeted rRNAsubsequences in particular organisms or groups of organisms in a samplehave been described previously. These highly specific probe sequencesadvantageously do not cross react with nucleic acids from any otherbacterial species or infectious agent under appropriate stringencyconditions.

The present invention provides polynucleotide probes that can be used todetect the Actinomycetes in a highly specific manner.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an oligonucleotide probethat specifically hybridizes under high stringency hybridizationconditions to a nucleic acid target region characteristic ofActinomycetes bacteria to form a detectable probe:target duplex. Thistarget region corresponds to E. coli rRNA nucleotide positions1986-2064. The invented oligonucleotide probe has a length of up to 100nucleotides and includes at least 17 contiguous nucleotides containedwithin the sequence of SEQ ID NO:10 or the complement thereof. Incertain embodiments, the oligonucleotide probe includes at least 25contiguous nucleotides, and may include at least 29 contiguousnucleotides contained within the sequence of SEQ ID NO:10. The highstringency hybridization condition may be provided either by 0.48 Msodium phosphate buffer, 0.1% sodium dodecyl sulfate and 1 mM each ofEDTA and EGTA, or by 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mMlithium succinate and 10 mM each of EDTA and EGTA. The oligonucleotideprobe may be made of DNA, but also may include at least one nucleotideanalog. For example, the probe may include at least one nucleotideanalog that has a methoxy group at the 2′ position of a ribose moiety.In one embodiment the invented oligonucleotide probe has a sequence thatis any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or thecomplement thereof, or SEQ ID NO:3 or the complement thereof. In apreferred embodiment, the sequence of the oligonucleotide is given bySEQ ID NO:2 or SEQ ID NO:3, and the oligonucleotide is a helperoligonucleotide. Any of the disclosed oligonucleotides can include adetectable label. Particular examples of detectable labels includechemiluminescent labels and radiolabels. In another preferredembodiment, the oligonucleotide has a sequence given by SEQ ID NO:1.This oligonucleotide is particularly useful as a hybridization probe,and may include a detectable label. A highly preferred detectable labelfor the hybridization probe is an acridinium ester.

Another aspect of the present invention relates to a probe compositionfor detecting nucleic acids of Actinomycetes bacteria. This compositionincludes an oligonucleotide probe that hybridizes under a highstringency condition to an Actinomycetes nucleic acid target regioncorresponding to E. coli 23S rRNA nucleotide positions 1986-2064 to forma detectable target:probe duplex. This oligonucleotide probe has alength of up to 100 nucleotide bases and includes at least 17 contiguousnucleotides contained within the sequence of SEQ ID NO: 10 or thecomplement thereof. Under high stringency hybridization conditions theoligonucleotide probe specifically hybridizes nucleic acids present inCorynebacterium aquaticum, Corynebacterium jeikieum, Corynebacteriumxerosis, Micrococcus luteus, Propionibacterium acnes, Mycobacteriumchelonae, Mycobacterium terrae, Mycobacterium intracellulare,Mycobacterium simiae, Mycobacterium avium, Mycobacterium scrofulaceum,Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium smegatis,Mycobacteriumfortuitum, Mycobacterium gastri, Mycobacterium xenopi,Mycobacterium marinum and Mycobacterium phlei. It is preferred that theoligonucleotide probe have a length of up to 100 nucleotide bases andinclude at least 25 contiguous nucleotides contained within the sequenceof SEQ ID NO:10 or the complement thereof. In certain embodiments, theinvented oligonucleotide probe is made of DNA. Examples of useful highstringency hybridization conditions are alternatively provided by 0.48 Msodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each ofEDTA and EGTA, or by 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mMlithium succinate and 10 mM each of EDTA and EGTA. In a highly preferredembodiment, the oligonucleotide probe has the sequence of SEQ ID NO:1 orthe complement thereof. In a preferred embodiment, the length of theoligonucleotide probe may be up to 60 bases. A highly preferredoligonucleotide probe has the length and sequence of SEQ ID NO:1.Certain embodiments of the invented probe composition include adetectable label on the oligonucleotide probe. For example, when theoligonucleotide probe has a length of up to 60 nucleotides, theoligonucleotide probe can include a detectable label. Alternatively,when the oligonucleotide probe has the sequence of SEQ ID NO:1 there canbe included a detectable label. Regardless of whether the probecomposition includes a labeled oligonucleotide probe of from 17-100nucleotides in length, or from 17-60 nucleotides in length, or havingthe sequence of SEQ ID NO: 1, the detectable label may be either achemiluminescent label (such as an acridinium ester) or a radiolabel. Itis preferred that these labeled oligonucleotide probes are used incombination with at least one helper oligonucleotide that facilitatesformation of the detectable probe:target duplex under high stringencyhybridization conditions. At least one of the helper oligonucleotidescan include at least one nucleotide analog. An example of a highlypreferred nucleotide analog would be one in which a ribose moiety has amethoxy group disposed at the 2′ position. In a highly preferredembodiment of the invention, the labeled oligonucleotide probe,regardless of its length, is used in combination with at least onehelper oligonucleotide having the sequence of SEQ ID NO:2 or SEQ IDNO:3.

Yet another aspect of the invention relates to a method for detectingthe presence of Actinomycetes bacteria in a test sample. This methodinvolves steps for providing to said test sample a probe compositioncomprising an oligonucleotide probe that hybridizes under a highstringency condition to an Actinomycetes nucleic acid target regioncorresponding to E. coli 23S rRNA nucleotide positions 1986-2064 to forma detectable target:probe duplex, said oligonucleotide probe having alength of up to 100 nucleotide bases and comprising at least 25contiguous nucleotides contained within the sequence of SEQ ID NO: 10 orthe complement thereof, and wherein under said hybridization conditionsaid oligonucleotide probe specifically hybridizes nucleic acids presentin Corynebacterium aquaticum, Corynebacterium jeikieum, Corynebacteriumxerosis, Micrococcus luteus, Propionibacterium acnes, Mycobacteriumchelonae, Mycobacterium terrae, Mycobacterium intracellulare,Mycobacterium simiae, Mycobacterium avium, Mycobacterium scrofulaceum,Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium smegatis,Mycobacterium fortuitum, Mycobacterium gastri, Mycobacterium xenopi,Mycobacterium marinum and Mycobacterium phlei. Thereafter, the resultingmixture is hybridized under high stringency conditions so that anyActinomycetes nucleic acid that may be present in the test samplecombines with the probe composition to form a probe:target duplex.Finally, the method involves detecting the probe:target duplex as anindicator of the presence of Actinomycetes bacteria in the test sample.In one embodiment of the invented method the test sample includesbacteria, and there is conducted a preliminary step for releasingnucleic acid from any bacteria that may be present in said test sample.In a different embodiment of the method the test sample is a lysate. Ingeneral, the high stringency hybridization conditions can be providedeither by: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecylsulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithiumlauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA.However, it is to be understood that other high stringency hybridizationconditions can give good results. In a preferred embodiment, theoligonucleotide probe used in the invented method has the length andsequence of SEQ ID NO:1. When this is the case, the oligonucleotideprobe may include a detectable label. In a highly preferred embodimentthe labeled oligonucleotide probe has the sequence of SEQ ID NO:1, thelabel on the probe is an acridinium ester, and the detecting step in theinvented method involves performing luminometry to detect probe:targetduplexs. In instances wherein the labeled oligonucleotide probe has thesequence of SEQ ID NO:1, the probe composition further may include atleast one helper oligonucleotide that facilitates formation ofprobe:target duplexs. Exemplary helper oligonucleotide have thesequences of SEQ ID NO:2 and SEQ ID NO:3.

Still yet another aspect of the invention relates to a kit that can beused for detecting the presence of Actinomycetes nucleic acids in a testsample. The kit contains a probe composition that includes anoligonucleotide probe that hybridizes under a high stringency conditionto an Actinomycetes nucleic acid target region corresponding to E. coli23S rRNA nucleotide positions 1986-2064 to form a detectabletarget:probe duplex. the oligonucleotide probe has a length of up to 100nucleotide bases and includes at least 25 contiguous nucleotidescontained within the sequence of SEQ ID NO:10 or the complement thereof.Under the high stringency hybridization condition the oligonucleotideprobe specifically hybridizes nucleic acids present in Corynebacteriumaquaticum, Corynebacterium jeikieum, Corynebacterium xerosis,Micrococcus luteus, Propionibacterium acnes, Mycobacterium chelonae,Mycobacterium terrae, Mycobacterium intracellulare, Mycobacteriumsimiae, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacteriumgordonae, Mycobacterium kansasii, Mycobacterium smegatis,Mycobacteriumfortuitum, Mycobacterium gastri, Mycobacterium xenopi,Mycobacterium marinum and Mycobacterium phlei. Additionally, theinvented kit includes printed instructions specifying, in order ofimplementation, the steps to be followed for detecting the Actinomycetesnucleic acid by detecting a complex between the oligonucleotide probeand an Actinomycetes nucleic acid target. Both the probe composition andthe printed instructions are in packaged combination with each other.

Definitions

As used herein, the following terms have the given meanings unlessexpressly stated to the contrary.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. Thesugar of a 5′-nucleotide contains a hydroxyl group (—OH) at the5′-carbon-5 position. The term also includes analogs of naturallyoccurring nucleotides and particularly includes analogs having a methoxygroup at the 2′ position of the ribose (OMe). As used herein, methoxyoligonucleotides containing “T” residues have a methoxy group at the 2′position of the ribose moiety, and a uracil at the base position of thenucleotide.

A “non-nucleotide unit” is a unit which does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

An “oligonucleotide” is a nucleotide polymer having two or morenucleotide subunits covalently joined together. Oligonucleotides aregenerally about 10 to about 100 nucleotides in length. The sugar groupsof the nucleotide subunits may be ribose, deoxyribose, or modifiedderivatives thereof such as OMe. The nucleotide subunits may by joinedby linkages such as phosphodiester linkages, modified linkages or bynon-nucleotide moieties that do not prevent hybridization of theoligonucleotide to its complementary target nucleotide sequence.Modified linkages include those in which a standard phosphodiesterlinkage is replaced with a different linkage, such as a phosphorothioatelinkage, a methylphosphonate linkage, or a neutral peptide linkage.Nitrogenous base analogs also may be components of oligonucleotides inaccordance with the invention.

A “target nucleic acid” is a nucleic acid comprising a target nucleicacid sequence.

A “target nucleic acid sequence,” “target nucleotide sequence” or“target sequence” is a specific deoxyribonucleotide or ribonucleotidesequence that can be hybridized by an oligonucleotide.

An “oligonucleotide probe” is an oligonucleotide having a nucleotidesequence sufficiently complementary to its target nucleic acid sequenceto be able to form a detectable hybrid probe:target duplex under highstringency hybridization conditions. An oligonucleotide probe is anisolated chemical species and may include additional nucleotides outsideof the targeted region as long as such nucleotides do not preventhybridization under high stringency hybridization conditions.Non-complementary sequences, such as promotor sequences, restrictionendonuclease recognition sites, or sequences that confer a desiredsecondary or tertiary structure such as a catalytic active site can beused to facilitate detection using the invented probes. Anoligonucleotide probe optionally may be labeled with a detectable moietysuch as a radioisotope, a fluorescent moiety, a chemiluminescent moiety,an enzyme or a ligand, which can be used to detect or confirm probehybridization to its target sequence. Oligonucleotide probes arepreferred to be in the size range of from 10 to 100 nucleotides inlength.

A “detectable moiety” is a molecule attached to, or synthesized as partof, a nucleic acid probe. This molecule should be uniquely detectableand will allow the probe to be detected as a result. These detectablemoieties are often radioisotopes, chemiluminescent molecules, enzymes,haptens, or even unique oligonucleotide sequences.

A “hybrid” or a “duplex” is a complex formed between two single-strandednucleic acid sequences by Watson-Crick base pairings or non-canonicalbase pairings between the complementary bases.

“Hybridization” is the process by which two complementary strands ofnucleic acid combine to form a double-stranded structure (“hybrid” or“duplex”). “Complementarity” is a property conferred by the basesequence of a single strand of DNA or RNA which may form a hybrid ordouble-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bondingbetween Watson-Crick base pairs on the respective strands. Adenine (A)ordinarily complements thymine (T) or uracil (U), while guanine (G)ordinarily complements cytosine (C).

“Mismatch” refers to any pairing, in a hybrid, of two nucleotides whichdo not form canonical Watson-Crick hydrogen bonds. In addition, for thepurposes of the following discussions, a mismatch can include aninsertion or deletion in one strand of the hybrid which results in anunpaired nucleotide(s).

The term “stringency” is used to describe the temperature and solventcomposition existing during hybridization and the subsequent processingsteps. Under high stringency conditions only highly complementarynucleic acid hybrids will form; hybrids without a sufficient degree ofcomplementarity will not form. Accordingly, the stringency of the assayconditions determines the amount of complementarity needed between twonucleic acid strands forming a hybrid. Stringency conditions are chosento maximize the difference in stability between the hybrid formed withthe target and the non-target nucleic acid. Exemplary high stringencyconditions are provided in the working Examples.

The term “probe specificity” refers to a characteristic of a probe whichdescribes its ability to distinguish between target and non-targetsequences.

The term “variable region” refers to a nucleotide polymer which differsby at least one base between the target organism and non-targetorganisms contained in a sample.

A “conserved region” is a nucleic acid subsequence which is not variablebetween at least two different polynucleotides.

“Bacteria” are members of the phylogenetic group eubacteria, which isconsidered one of the three primary kingdoms.

The term “sequence divergence” refers to a process by which nucleotidepolymers become less similar during evolution.

The term “sequence convergence” refers to a process by which nucleotidepolymers become more similar during evolution.

“Tm” refers to the temperature at which 50% of the probe is convertedfrom the hybridized to the unhybridized form.

A “helper oligonucleotide” is an oligonucleotide that binds a region ofa target nucleic acid other than the region that is bound by anoligonucleotide probe. Helper oligonucleotides impose new secondary andtertiary structures on the targeted region of the single-strandednucleic acid so that the rate of binding of the oligonucleotide probe isaccelerated. Although helper oligonucleotides are not labeled with adetectable label when used in conjunction with labeled oligonucleotideprobes, they facilitate binding of labeled probes and so indirectlyenhance hybridization signals.

The phrases “consist essentially of” or “consisting essentially of”means that the oligonucleotide has a nucleotide sequence substantiallysimilar to a specified nucleotide sequence. Any additions or deletionsare non-material variations of the specified nucleotide sequence whichdo not prevent the oligonucleotide from having its claimed property,such as being able to preferentially hybridize under high stringencyhybridization conditions to its target nucleic acid over non-targetnucleic acids.

One skilled in the art will understand that substantially correspondingprobes of the invention can vary from the referred-to sequence and stillhybridize to the same target nucleic acid sequence. This variation fromthe nucleic acid may be stated in terms of a percentage of identicalbases within the sequence or the percentage of perfectly complementarybases between the probe and its target sequence. Probes of the presentinvention substantially correspond to a nucleic acid sequence if thesepercentages are from 100% to 80% or from 0 base mismatches in a 10nucleotide target sequence to 2 bases mismatched in a 10 nucleotidetarget sequence. In preferred embodiments, the percentage is from 100%to 85%. In more preferred embodiments, this percentage is from 90% to100%; in other preferred embodiments, this percentage is from 95% to100%.

By “sufficiently complementary” or “substantially complementary” ismeant nucleic acids having a sufficient amount of contiguouscomplementary nucleotides to form, under high stringency hybridizationconditions, a hybrid that is stable for detection.

By “nucleic acid hybrid or “probe:target duplex” is meant a structurethat is a double-stranded, hydrogen-bonded structure, preferably 10 to100 nucleotides in length, more preferably 14 to 50 nucleotides inlength. The structure is sufficiently stable to be detected by meanssuch as chemiluminescent or fluorescent light detection,autoradiography, electrochemical analysis or gel electrophoresis. Suchhybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

By “negative sense” is meant a nucleic acid molecule perfectlycomplementary to a reference (i.e., sense) nucleic acid molecule.

“RNA and DNA equivalents” refer to RNA and DNA molecules having the samecomplementary base pair hybridization properties. RNA and DNAequivalents have different sugar groups (i.e., ribose versusdeoxyribose), and may differ by the presence of uracil in RNA andthymine in DNA. The difference between RNA and DNA equivalents do notcontribute to differences in substantially corresponding nucleic acidsequences because the equivalents have the same degree ofcomplementarity to a particular sequence.

By “preferentially hybridize” is meant that under high stringencyhybridization conditions oligonucleotide probes can hybridize theirtarget nucleic acids to form stable probe:target hybrids (therebyindicating the presence of the target nucleic acids) without formingstable probe:non-target hybrids (that would indicate the presence ofnon-target nucleic acids from other organisms). Thus, the probehybridizes to target nucleic acid to a sufficiently greater extent thanto non-target nucleic acid to enable one skilled in the art toaccurately detect the presence of Actinomycetes bacteria and distinguishtheir presence from that of other organisms. Preferential hybridizationcan be measured using techniques known in the art and described herein.For example, when compared with hybridization to C. albicans nucleicacids, oligonucleotide probes of the invention preferentially hybridizeActinomycetes nucleic acids by about 100-2,000 fold.

An Actinomycetes “target nucleic acid sequence region” refers to anucleic acid sequence present in the nucleic acid of Actinomycetesbacteria or a sequence complementary thereto, which is not present inthe nucleic acids of other species. Nucleic acids having nucleotidesequences complementary to a target sequence may be generated by targetamplification techniques such as polymerase chain reaction (PCR) ortranscription mediated amplification (e.g., Kacian and Fultz, NucleicAcid Sequence Amplification Methods, U.S. Pat. No. 5,824,518).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of one oligonucleotide probe and two helper Ifoligonucleotides with a collection of positively reactive andnon-reactive target sequences.

DETAILED DESCRIPTION OF THE INVENTION

Herein we disclose preferred target nucleotide sequences foroligonucleotide probes and helper oligonucleotides that can be used todetect and identify rRNA or rDNA of Actinomycetes bacteria. Highlypreferred polynucleotide probes and accessory helper oligonucleotidesthat are useful for specifically detecting Actinomycetes areparticularly disclosed. The probes, which are complementary toparticular rRNA sequences of the 23S rRNA, advantageously are capable ofdistinguishing Actinomycetes from the known phylogenetically nearestneighbors.

In addition to having nucleic acid sequences that permit hybridizationto the ribosomal RNA (rRNA) or DNA (rDNA) sequences of Actinomycetesbacteria, the oligonucleotide probes of the invention are at least 90%complementary, preferably perfectly complementary, to at least a portionof the described target sequence region identified by SEQ ID NO:11. Theportion is preferably at least 17 nucleotides in length, more preferablyat least 25 nucleotides in length, and still more preferably at least 29nucleotides in length.

As indicated above, the invented oligonucleotides are targeted tonucleic acid sequences of Actinomycetes bacteria. These oligonucleotidescan be used as probes that preferentially hybridize to an Actinomycetesnucleic acid target region to form a detectable duplex that indicatesthe presence of Actinomycetes bacteria. Alternatively, the inventedoligonucleotides can be used as Helper oligonucleotides that hybridizeto an Actinomycetes nucleic acid target region under high stringencyhybridization conditions, and that can enhance the formation of a duplexbetween a labeled oligonucleotide probe and its complementary targetnucleic acid.

In preferred embodiments, the oligonucleotide probes described hereinselectively hybridize nucleic acids from Actinomycetes bacteria overthose from other organisms under high stringency hybridizationconditions. In some embodiments of the present invention, theoligonucleotide probe comprises a detectable moiety, such as anacridinium ester or a radioisotope.

Preferred methods for detecting the presence of Actinomycetes bacteriainclude the step of contacting a test sample under high stringencyhybridization conditions with an oligonucleotide probe thatpreferentially hybridizes to an Actinomycetes target nucleic acidsequence over a nucleic acid sequence of other organisms. Preferably thetarget nucleic acid sequence is fully complementary to a sequence of atleast 17 contiguous nucleotides, more preferably at least 25 contiguousnucleotides, still more preferably at least 29 contiguous nucleotidescontained in the sequence given by SEQ ID NO:11. Thus, oligonucleotidesuseful in connection with the invention preferably are up to 100nucleotides in length and have at least 17 contiguous nucleotides, morepreferably at least 25 contiguous nucleotides, still more preferably atleast 29 contiguous nucleotides contained in the sequence given byGGTCTTTCCGTCCTGCCGCGCGTAACGAGCATCTrTACTCGTAGTGCAATTlCGCCGAGTCTGTGGTTGAGACAGTGGG (SEQ ID NO:10) or the complement thereof.The oligonucleotides may be RNA and DNA equivalents, and may containnucleotide analogs.

Introduction and Background

In the development of the invention, rRNA sequences from a collection ofrelated and unrelated organisms were aligned to identify candidateconserved sequences present in the 23S rRNA that could be used todistinguish Actinomycetes organisms from other bacterial as well aseukaryotic organisms. The rRNA or rDNA sequences of Actinomycetes anddistant phylogenetic neighbors were aligned to reveal areas of maximumhomology. Homologous regions were examined for sequence variation inorder to identify rRNA sequences that were conserved among theActinomycetes and that showed mismatches with other closely anddistantly related genera. The sequences deduced as candidate probesaccording to the methods described below finally were tested against apanel of rRNA standards and bacterial lysates to verify their utility asprobes under laboratory conditions.

Polynucleotide sequences of rRNAs are most conveniently determined usinga dideoxynucleotide sequencing procedure. In this procedure,oligonucleotide primers of about 10-100 bases in length andcomplementary to conserved regions of rRNA from any of the 5S, 16S or23S ribosome subunits can be extended by reverse transcriptase. Theresulting DNA extension products can then be sequenced either bychemical degradation or by dideoxynucleotide sequencing (Lane et al.,Proc. Natl. Acad. Sci. USA 82: 6955 (1985)). According to anotherpreferred method, genomic sequences encoding the rRNA can also bedetermined.

The strong interdependence of secondary structure and function of therRNA molecules is well known. Indeed, evolutionary changes in theprimary sequence of the rRNA are effectively restricted such thatsecondary structure of the molecule will be maintained. For example, ifa base is changed on one side of a helix of a rRNA molecule, then acompensating change will be made on the other side of the helix topreserve complementarity (this is referred to as covariance). Thisrelationship allows two very different rRNA sequences to be “aligned”based on conserved primary sequence and conserved elements of thesecondary structure. Once the sequences have been aligned, it becomespossible to identify conserved and variable regions of the rRNAsequence.

Variable regions of rRNAs were identified by comparative analysis usingpublished rRNA sequences and sequences that were determined during thedevelopment of the present invention. Commercially available softwarecan be used or adapted for the purposes disclosed herein. Since thesequence evolution at each of the variable regions (for example,spanning a minimum of 10 nucleotides) of rRNA is, for the most part,divergent and not convergent, we can confidently design probes based ona few rRNA sequences which differ between the target organism and itsphylogenetically closest relatives. Indeed, we have detected sufficientvariation between the rRNA sequences of numerous target organisms andtheir closest phylogenetic relatives in a single sample to permit thedesign of a probe that can be used according to the methods describedbelow.

Probe Selection Guidelines

The following general guidelines can be used for designing probes havingdesirable characteristics in accordance with the present invention.Manipulation of one or more of the many factors that influence theextent and specificity of a hybridization reaction can determine thesensitivity and specificity of a particular probe. This is true whetheror not the probe is perfectly complementary over the full length of itstarget polynucleotide sequence. Guidelines for preparing probes usefulin connection with the invention now follow.

First, the stability of the probe:target nucleic acid hybrid should bechosen to be compatible with the assay conditions. This may beaccomplished by avoiding long A and T rich sequences, by terminating thehybrids with G:C base pairs and by designing the probe in such a waythat the Tm will be appropriate for standard conditions to be employedin the assay. The nucleotide sequence of the probe should be chosen sothat the length and %G and %C result in a probe having a Tm about 2-10°C. higher than the temperature at which the final assay will beperformed. The base composition of the probe is significant because G:Cbase pairs exhibit greater thermal stability when compared with A:T orA:U base pairs. Thus, hybrids involving complementary nucleic acidshaving a high G:C content will be stable at higher temperatures whencompared with hybrids having a lower G:C content.

Ionic strength and temperature conditions at which a hybridizationreaction will be conducted also should be considered when designing aprobe having a negatively charged backbone, such as would be provided byphosphodiester linkages between nucleotides. It is generally known thathybridization rate increases as ionic strength of the reaction mixtureincreases. Similarly, the thermal stability of hybrids increases withincreasing ionic strength. Conversely, hydrogen bond-disrupting reagentssuch as formamide, urea, DMSO and alcohols increase the stringency ofhybridization. Destabilization of hydrogen bonds by reagents in thisclass can greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Hybridization reactions conducted below the temperature optimum mayallow mismatched base sequences to hybridize and can result in reducedprobe specificity.

Second, the position at which the probe binds its target polynucleotideshould be chosen to minimize the stability of hybrids formed betweenprobe:non-target polynucleotides. This may be accomplished by minimizingthe length of perfect complementarity with polynucleotides of non-targetorganisms, by avoiding G:C rich regions of homology with non-targetsequences, and by positioning the probe to span as many destabilizingmismatches as possible. Whether a probe sequence will be useful fordetecting only a specific type of organism depends largely on thermalstability differences between probe:target hybrids and probe:non-targethybrids. The differences in Tm should be as large as possible to producehighly specific probes.

The length of the target nucleic acid sequence and the correspondinglength of the probe sequence also are important factors to be consideredwhen designing a probe useful for specifically detecting Actinomycetes.While it is possible for polynucleotides that are not perfectlycomplementary to hybridize to each other, the longest stretch ofperfectly homologous base sequence will ordinarily be the primarydeterminant of hybrid stability.

Third, regions of the rRNA which are known to form strong internalstructures inhibitory to hybridization of a probe are less preferred astargets. Probes having extensive self-complementarity also should beavoided. As indicated above, hybridization is the association of twosingle strands of complementary nucleic acid to form a hydrogen bondeddouble-stranded structure. If one of the two strands is wholly orpartially double-stranded, then it will be less able to participate inthe formation of a new hybrid. Significantly, all rRNA molecules formvery stable intramolecular hybrids.

The rate and extent of hybridization between a probe and its target canbe increased substantially by designing the probe such that asubstantial portion of the sequence of interest is single-stranded. Ifthe target nucleic acid to be detected is a genomic sequence encoding arRNA, then that target will naturally occur in a double-stranded form.This is also the case with products of the polymerase chain reaction(PCR). These double-stranded targets are naturally inhibitory tohybridization with a probe. Finally, undesirable intramolecular andintermolecular hybrids can form within a single probe molecule orbetween different probe molecules if there is sufficientself-complementarity. Thus, extensive self-complementarity in a probesequence should be avoided.

Preferably, probes useful for carrying out the procedures describedbelow will hybridize only under conditions of high stringency. Underthese conditions only highly complementary nucleic acid hybrids willform (i.e., those having at least 14 out of 17 bases in a contiguousseries of bases being complementary). Hybrids will not form in theabsence of a sufficient degree of complementarity. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. Stringency is chosen to maximize the difference in stabilitybetween the hybrid formed with the target and non-target nucleic acid.Exemplary high stringency conditions are employed in the Examplespresented below.

While oligonucleotide probes of different lengths and base compositionmay be used for detecting Actinomycetes, preferred probes in thisinvention have lengths of up to 100 nucleotides, and more preferably 60nucleotides. Preferred length ranges for the invented oligonucleotidesare from 10 to 100 bases in length, or more preferably between 15 and 50bases in length, and are sufficiently homologous to the target nucleicacid to permit hybridization under high stringency conditions such asthose employed in the Examples described below. However, the specificprobe sequences described below also may be provided in a nucleic acidcloning vector or transcript or other longer nucleic acid and still canbe used for detecting members of the Actinomycetes.

Chemical Structure of Oligonucleotides

All of the oligonucleotides of the present invention may be modifiedwith chemical groups to enhance their performance. Thus, it is to beunderstood that references to “oligonucleotide probes” or “helperoligonucleotides” or simply “oligonucleotides” embrace polymers ofnative nucleotides as well as polymers that include at least onenucleotide analog.

Backbone-modified oligonucleotides, such as those havingphosphorothioate or methylphosphonate groups, are examples of analogsthat can be used in conjunction with oligonucleotides of the presentinvention. These modifications render the oligonucleotides resistant tothe nucleolytic activity of certain polymerases or to nuclease enzymes.Other analogs that can be incorporated into the structures of theoligonucleotides disclosed herein include peptide nucleic acids, or“PNAs.” The PNAs are compounds comprising ligands linked to a peptidebackbone rather than to a phosphodiester backbone. Representativeligands include either the four main naturally occurring DNA bases(i.e., thymine, cytosine, adenine or guanine) or other naturallyoccurring nucleobases (e.g., inosine, uracil, 5-methylcytosine orthiouracil) or artificial bases (e.g., bromothymine, azaadenines orazaguanines, etc.) attached to a peptide backbone through a suitablelinker. The PNAs are able to bind complementary ssDNA and RNA strands.Methods for making and using PNAs are disclosed in U.S. Pat. No.5,539,082. Another type of modification that can be used to makeoligonucleotides having the sequences described herein involves the useof non-nucleotide linkers (e.g., Arnold, et al., “Non-Nucleotide LinkingReagents for Nucleotide Probes”, U.S. Pat. No. 6,031,091 herebyincorporated by reference) incorporated between nucleotides in thenucleic acid chain which do not interfere with hybridization or theelongation of a primer.

Nucleic Acid Based Methods of Detecting rRNA or rDNA

A composition that includes an oligonucleotide probe, either alone or incombination with one or more helper oligonucleotides, can be used fordetecting rRNA or rDNA of Actinomycetes bacteria in a hybridizationassay. Defined oligonucleotides that can be used to practice theinvention can be produced by any of several well-known methods,including automated solid-phase chemical synthesis usingcyanoethylphosphoramidite precursors (Barone et al., Nucl Acids Res12:4051 (1984)). Other well-known methods for preparing syntheticoligonucleotides also may be employed.

Essentially any labeling and detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction with the probes disclosed herein when a labeled probe isdesired. Included among the collection of useful labels are: isotopiclabels, enzymes, haptens, linked oligonucleotides, chemiluminescentmolecules and redox-active moieties that are amenable to electrochemicaldetection methods. Standard isotopic labels that can be used to producelabeled oligonucleotides include ³H, ³⁵S, ³²p, ¹²⁵I, ⁵⁷Co and ¹⁴C. Whenusing radiolabeled probes, hybrids can be detected by autoradiography,scintillation counting or gamma counting.

Non-isotopic materials can also be used for labeling oligonucleotideprobes. These non-isotopic labels can be positioned internally or at aterminus of the oligonucleotide probe. Modified nucleotides may beincorporated enzymatically or chemically with modifications of the probebeing performed during or after probe synthesis, for example, by the useof non-nucleotide linker groups. Non-isotopic labels include fluorescentmolecules, chemiluminescent molecules, enzymes, cofactors, enzymesubstrates, haptens or other ligands. Acridinium esters are particularlypreferred non-isotopic labels for detecting probe hybrids.

Indeed, any number of different non-isotopic labels can be used forpreparing labeled oligonucleotides in accordance with the invention.Preferred chemiluminescent molecules include acridinium esters of thetype disclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use inconnection with homogenous protection assays, and of the type disclosedby Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connectionwith assays that quantify multiple targets in a single reaction. Thedisclosures contained in these patent documents are hereby incorporatedby reference. U.S. Pat. No. 5,998,135 discloses yet another method thatcan be used for labeling and detecting the probes of the presentinvention using fluorimetry to detect fluorescence emission fromlanthanide metal labels disposed on probes, where the emission fromthese labels becomes enhanced when it is in close proximity to an energytransfer partner. Preferred electrochemical labeling and detectionapproaches are disclosed in U.S. Pat. Nos. 5,591,578 and 5,770,369, andthe published International Patent Application No. PCT/US98/12082, thedisclosures of which are hereby incorporated by reference. Redox activemoieties useful as electrochemical labels in the present inventioninclude transition metals such as Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru.

Those having an ordinary level of skill in the art will appreciate thatalternative procedures for detecting nucleic acids of Actinomycetesbacteria using the invented probes can be carried out using eitherlabeled probes or unlabeled probes. For example, hybridization assaymethods that do not rely on the use of a labeled probe are disclosed inU.S. Pat. No. 5,945,286 which describes immobilization of unlabeledprobes made of peptide nucleic acids (PNAs), and detectably labeledintercalating molecules which can bind double-stranded PNA probe/targetnucleic acid duplexes. In these procedures, as well as in certainelectrochemical detection procedures, such as those disclosed inpublished International Patent Application No. PCT/US98/12082 entitled“Detection of Analytes Using Reorganization Energy,” publishedInternational Patent Application No. PCT/US98/12430 entitled “ElectronicMethods for the Detection of Analytes,” and in published InternationalPatent Application No. PCT/US97/20014 entitled “Electrodes Linked ViaConductive Oligomers to Nucleic Acids” the oligonucleotide probe is notrequired to harbor a detectable label.

Acceptability of the final product following synthesis and purificationof an oligonucleotide may be verified by any of several procedures.First, polyacrylamide gel electrophoresis can be used to determine thesize and purity of the oligonucleotide according to standard laboratorymethods (see Molecular Cloning: A Laboratory Manual, Sambrook et al.,eds. Cold Spring Harbor Lab Publ., 11.51, (1989)). Alternatively, HighPressure Liquid Chromatography (“HPLC”) procedures can be used for thissame purpose.

Hybridization between the labeled oligonucleotide probe and targetnucleic acid in the procedures described below can be enhanced throughthe use of unlabeled “helper oligonucleotides” according to theprocedure disclosed by Hogan et al., in U.S. Pat. No. 5,030,557entitled, “Means and Methods for Enhancing Nucleic Acid Hybridization.”As indicated above, helper oligonucleotides bind a region of the targetnucleic acid other than the region that is bound by the assay probe.This binding imposes new secondary and tertiary structures on thetargeted region of the single- stranded nucleic acid and accelerates therate of probe binding. Helper oligonucleotides which can be used incombination with labeled oligonucleotide probes of the present inventionare preferably 17 to 100 nucleotides in length and have a sequence thatincludes at least 17 contiguous nucleotides contained within thesequence of SEQ ID NO: 10. Other preferred helper oligonucleotides havelengths of up to 100 nucleotides and include at least 25 contiguousnucleotides contained within the sequence of SEQ ID NO: 10.

Those having ordinary skill in the art will appreciate that factorsaffecting the thermal stability of a probe:target hybrid also caninfluence probe specificity. Accordingly, the melting profile, includingthe melting temperature (Tm) of probe:target hybrids, should beempirically determined for each probe:target combination. A preferredmethod for making this determination is described by Arnold et al., inU.S. Pat. No. 5,283,174, entitled “Homogeneous Protection Assay.”

One approach for measuring the Tm of a probe:target hybrid involvesconducting a hybridization protection assay. According to the method ofthis assay, a probe:target hybrid is formed under conditions of targetexcess in a lithium succinate buffered solution containing lithiumlauryl sulfate. Aliquots of the “preformed” hybrids are diluted in thehybridization buffer and incubated for five minutes at varioustemperatures starting below the anticipated Tm (typically 55° C.) andincreasing in 2-5 degree increments. This solution is then diluted witha mildly alkaline borate buffer and incubated at a lower temperature(for example 50° C.) for ten minutes. An acridinium ester (AE) linked toa single-stranded probe will be hydrolyzed under these conditions whilean acridinium ester linked to a hybridized probe will be relatively“protected.” This procedure is referred to as the hybridizationprotection assay (“HPA”). The amount of chemiluminescence remaining isproportional to the amount of hybrid and is measured in a luminometer byaddition of hydrogen peroxide followed by alkali. The data is plotted aspercent of maximum signal (usually from the lowest temperature) versustemperature. The Tm is defined as the point at which 50% of the maximumsignal remains.

In an alternative approach, the Tm of a probe:target hybrid can bedetermined using an isotopically labeled probe. In all cases, the Tm fora given hybrid will vary depending on the concentration of salts,detergents and other solutes contained in the hybridization solution.All of these factors influence relative hybrid stability during thermaldenaturation (Molecular Cloning: A Laboratory Manual Sambrook et al.,eds. Cold Spring Harbor Lab Publ., 9.51 (1989)).

The rate at which a probe hybridizes to its target is a measure of thethermal stability of the target secondary structure in the probe region,and can be determined using C₀t_(½) measurements. These kineticmeasurements of hybridization rate have units of (moles of nucleotideper liter)×(seconds). Expressed more simply, the C₀t_(½) value is theconcentration of probe times the half-life of hybridization at thatconcentration. This value can be determined by hybridizing variousamounts of probe to a constant amount of target nucleic acid for a fixedtime. For example, 0.05 pmol of target is incubated with 0.012, 0.025,0.05, 0.1 and 0.2 pmol of probe for 30 minutes. The C₀t_(½) may also bedetermined by hybridizing the target and probe under conditions oftarget excess and then measuring the increase of duplex formation overtime. The amount of hybrid present can be measured using theabove-described HPA procedure or by scintillation counting if aradiolabeled probed is used in the procedure. The measured signal, whenusing AE labeled probe, is then plotted as the log of the percent ofmaximum Relative Light Units (“RLU”) from the highest probeconcentration versus probe concentration (moles of nucleotide perliter). The C₀t_(½) is graphically determined from the concentrationcorresponding to 50% of maximum hybridization multiplied by thehybridization time in seconds. These values range from 9×10⁻to 9×10⁻⁵with the preferred values being less than 3.5×10⁻⁵. Similar values maybe obtained by measuring radioactivity and plotting % hybridization at agiven time point vs maximum extent.

In a preferred method of determining whether a biological samplecontains rRNA or rDNA that would indicate the presence of members of theActinomycetes, nucleic acids may be released from bacterial cells bysonic disruption, for example according to the method disclosed byMurphy et al., in U.S. Pat. No. 5,374,522. Other known methods fordisrupting cells include the use of enzymes, osmotic shock, chemicaltreatment, and vortexing with glass beads. Other methods suitable forliberating from microorganisms the nucleic acids that can be subjectedto the hybridization methods disclosed herein have been described byClark et al., in U.S. Pat. No. 5,837,452 and by Kacian et al., in U.S.Pat. No. 5,5,364,763. Following or concurrent with the release of rRNA,labeled probe may be added in the presence of accelerating agents andincubated at the optimal hybridization temperature for a period of timenecessary to achieve significant hybridization reaction.

The following polynucleotide sequence was characterized by the criteriaof length, Tm and nucleotide sequence and was found to be specific forthe rRNA of Actinomycetes, CGAGCATCTTTACTCGTAGTGCAATTTCG (SEQ ID NO:1).This polynucleotide, referred to herein as MtuB2011, is complementary toa unique segment found in the 23S rRNA of all Actinomycetes. Arepresentative list of Actinomycetes bacteria can be found in Table 2.The probe is 29 bases in length, has an RXL linker between 11 and 12nucleotides from the 5′ end and has a Tm of 65.5° C., and hybridizedrRNA of Mycobacterium avium in a region corresponding to bases 2011-2040of E. coli 23S rRNA.

This probe is one illustration of an oligonucleotide that: (1)hybridizes the target nucleic acid under high stringency hybridizationconditions, (2) has a length of up to 100 nucleotide bases, and (3)includes at least 17 contiguous nucleotides falling within the 1986-2064target region identified by SEQ ID NO:10 or its complement. Otheroligonucleotides having these properties are contemplated for use ashybridization assay detection probes and are embraced by the invention.

Similarly, oligonucleotides having the sequences of SEQ ID NOs:2 and 3are disclosed herein as illustrations of useful helper oligonucleotides.Like the helper oligonucleotides employed in the working Examplesherein, other helper oligonucleotides embraced by the invention alsohave sequences of up to 100 nucleotides in length and further have atleast 17, or more preferably at least 25, contiguous nucleotidescontained within the target region identified by SEQ ID NO:10 or itscomplement.

As indicated below, the MtuB2011 probe hybridized Actinomycetes rRNA ina manner that was promoted by the use of helper oligonucleotides.According to the procedure used to make this determination,single-stranded probe oligonucleotide radiolabeled at the 5′-end wascontacted with rRNA from Mycobacterium avium in the presence or absenceof helper oligonucleotides. Probe molecules hybridizing the rRNA to formdouble-stranded hybrids were separated from single-stranded probemolecules by hydroxyapatite capture. The double-stranded hybrids boundto the hydroxyapatite and were detected and quantitated by scintillationcounting. The extent of hybridization was then calculated as apercentage. As indicated below, the Tm of the probe:target hybridadvantageously was significantly increased in the presence of one ormore helper oligonucleotides.

The following Example describes the methods used to demonstrate that theMtuB2011 probe hybridized rRNA from Mycobacterium avium and that thisinteraction was facilitated by including helper oligonucleotides in thehybridization mixture.

EXAMPLE 1 Tm Determination for Probe:Target Hybrids

Tm values for probe:target and helper:target hybrids were determinedusing an end-labeled probe having the sequence of MtuB2011 andend-labeled helper oligonucleotides selected from the group: (A)OMeFraB1986 and (B) OMeFraB2040. The sequence of MtuB2011 isCGAGCATCTTTACTCGTAGTGCAATTTCG (SEQ ID NO:1), the sequence of OMeFraB1986is CCGAGUCUGUGGUUGAGACAGUGGG (SEQ ID NO:2) and the sequence ofOMeFraB2040 is GGUCUUUCCGUCCUGCCGCGCGUAA (SEQ ID NO:3). Helperoligonucleotides A and B were selected to bind the rRNA of Actinomycetesin regions of the molecule immediately adjacent to the probe, helper Abinding in about the 1986-2010 region of the 23S rRNA, and helper Bbinding in the 2040-2064 region of the 23S rRNA. The probe and helperoligonucleotides were 5′-end labeled using [γ-³²P]ATP as a phosphatedonor and T4 polynucleotide kinase to catalyze the phosphate transferreaction essentially as described in Molecular Cloning: A LaboratoryManual (Sambrook et al., eds. Cold Spring Harbor Lab Publ. 10.59(1989)). End-labeled helper oligonucleotides were separately combinedwith purified rRNA from Mycobacterium gordonae and the probeoligonucleotide was combined with purified rRNA from Mycobacterium aviumto provide conditions of target excess. In trials that included both theprobe and helper oligonucleotides, only the probe was end-labeled andeach helper oligonucleotide was present in at least a 10 fold molarexcess over the Mycobacterium avium rRNA that served as a target. Allmixtures were hybridized to completion in a solution that included 0.48M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, 1 mM EDTA and 1mM EGTA. As negative controls, the probe and/or helper oligonucleotideswere hybridized in the absence of the nucleic acid target. At theconclusion of the hybridization procedure, mixtures were diluted andpassed over a hydroxyapatite column to separate single-stranded nucleicacids from double-stranded hybrids. The amount of radioactivity in thecolumn flow-through represented single- stranded probe and was measuredby scintillation counting. The amount of radioactivity bound to thehydroxyapatite was separately measured by scintillation counting. Theextent of hybrid formation, expressed as a percentage, was calculated bydividing the amount of probe (measured in cpm) bound to thehydroxyapatite by the total amount of probe (in cpm) that was applied tothe column. Results of these procedures are presented in Table 1.

TABLE 1 Hybridization of the MtuB2011 Probe with Target rRNAOligonucleotide(s) % Hybridization Tm (° C.) MtuB2011 (Probe) 84 65.5helper A, OMeFra1986 96 78 helper B, OMeFraB2040 96 ≈80 Probe + helper A98 68 Probe + helper B 97 67 Probe + helper A + helper B 96 69.5

The results from this procedure confirmed that the end-labeled probehybridized rRNA from Mycobacterium avium and/or Mycobacterium gordonaeand indicated this interaction advantageously was facilitated by helperoligonucleotides. We particularly observed that the Tm of theprobe:target complex could be increased from 65.5 to 69.5° C. when bothhelper oligonucleotides were included in the hybridization reaction.Although the probe can be used either alone or in combination with oneor more helper oligonucleotides for hybridizing Actinomycetes rRNA, thebelow-described experiments to characterize the probe were conductedusing the probe in combination with helper oligonucleotides having thesequences of OMeFraB 1986 and OMeFraB2040. Combinations of probe andhelper oligonucleotides useful in the procedures described hereinpreferably have probe:target Tm values in the range of from about 65-70°C. under the conditions described above.

Probe specificity was confirmed by demonstrating positive hybridizationto rRNAs from a specificity panel. The collection of organisms used assources of target nucleic acids in this procedure represented a broadtaxonomic cross-section of organisms and a nearest-neighbor group. Inthe following procedure, quantitative results using the AE-labeledhybridization probe were compared to the amount of bacterial rRNApresent in each sample using a positive control probe. This positivecontrol probe, which hybridized rRNA from all species of bacteria, wasparticularly useful for confirming the presence of bacterial rRNA insamples that failed to hybridize the MtuB201 1 probe. In such an event,the positive control probe provided confirmation for the presence ofhybridizable rRNA and so validated the negative results. In the case offungal rRNA targets, a broadly reactive fungal rRNA hybridization probeserved as the positive control.

The following Example describes the methods used to demonstrate that theMtuB2011 probe hybridized rRNAs from a panel of Actinomycetes organisms.

EXAMPLE 2 Verification of Probe Specificity

Bacterial lysates or purified RNA were used as nucleic acid targets forhybridization of a probe having the sequence ofCGAGCATCTTTACTCGTAGTGCAATTTCG (MtuB2011) (SEQ ID NO:1) together withhelper oligonucleotides having the sequences ofCCGAGUCUGUGGUUGAGACAGUGGG (OMeFraB1986) (SEQ ID NO:2) andGGUCUUUCCGUCCUGCCGCGCGUAA (OMeFraB2040) (SEQ ID NO:3). Organismsemployed as sources of rRNA in this procedure were either typed clinicalisolates or obtained from the American Type Culture Collection (ATCC).All samples are identified in Table 2 by master log numbers forGen-Probe Incorporated. Most samples were obtained from the ATCC.Parallel samples of each rRNA were hybridized with a labeled positivecontrol probe having the sequence CGACAAGGAAUUUCGC (OMe Eco B 1933) (SEQID NO:4) and unlabeled helper oligonucleotides having the sequencesUACCWUAGGACCGUUAU (OMe Eco B 1916) (SEQ ID NO:5) and CAGGUCGGAACUUACC(OMe Eco B 1949a) (SEQ ID NO:6). The hybridization solution contained0.6M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mMeach of EDTA and EGTA, pH 5.5. Both the MtuB2011 probe and the positivecontrol probe were labeled with acridinium ester essentially accordingto the method disclosed in U.S. Pat. No. 5,185,439, entitled “AcridiniumEster Labeling and Purification of Nucleotide Probes.” At the conclusionof the hybridization reaction, acridinium ester linked to unhybridizedprobe was rendered non-chemiluminescent under mild alkaline conditions,while acridinium ester attached to hybridized probe remained resistantto the inactivation. Conditions for the hydrolysis and detection ofhybridized probe labeled with acridinium ester are described by Arnoldet al., in Clin. Chem. 35:1588 (1989)). The magnitudes of probehybridization in these procedures were quantitated by luminometry usingprocedures familiar to those having ordinary skill in the art. Themagnitude of the Actinomycetes probe signal was then divided by themagnitude of the bacterial positive control signal to quantitativelynormalize results in the study. Samples having MtuB2011 probe signalsthat were greater than 30% of the positive control signal indicatedspecific hybridization with the MtuB2011 probe with helpers, while lowervalues indicated negative results for the assay fornat. Assay resultsare shown in Table 2.

TABLE 2 Hybridization of the MtuB2011 Probe and rRNA-Containing Lysatesfrom a Collection of Actinomycetes Hybridization Results rRNA SourcePan-Bacterial Actinomycetes Fractional *GP # (Organism) Probe (RLU)Probe (RLU) Hybridization (%) 236 Corynebacterium aquaticum 19450 1895097 239 Corynebacterium jeikieum 9258 4404 48 237 Corynebacterium xerosis9207 9624 105 240 Micrococcus luteus 2143 2301 107 190 Propionibacteriumacnes 5680 4804 85 159 Mycobacterium chelonae 256985 106195 41 543Mycobacterium terrae 43323 58933 136  87 Mycobacterium intracellulare80497 123777 154  84 Mycobacterium simiae 29865 39705 133 PN 101676Mycobacterium avium 1690 1551 92  79 Mycobacterium scrofulaceum 760110373 136  85 Mycobacterium fordon ae 22560 22180 98  82 Mycobacteriumkansasii 35457 44997 127  86 Nycobacterium smegatis 108778 137648 127160 Mycobacterium fortuitum 71278 127108 178  15 Mycobacterium gastri5406 7274 135  74 Mycobacterium xenopi 36080 27110 75  83 Mycobacteriummarinum 268900 330700 123  78 Mycobacterium phlei 12999 19780 152 *“GP#”entries indicate master log numbers for Gen-Probe Incorporated.

The results presented in Table 2 confirmed that the probe directedagainst Actinomycetes rRNA efficiently hybridized rRNA samples fromnumerous Actinomycetes species.

Specificity of the Actinomycetes-specific probe was further investigatedby hybridizing labeled probe with a collection of species representing abroad spectrum of phylogentically diverse organisms. In this procedure,AE-labeled probe was separately mixed with individual rRNA containinglysates from organisms that were only distant phylogenetic relatives ofthe Actinomycetes. Positive hybridization results obtained using thepositive control probe and negative results obtained using the MtuB2011probe in the following procedure further indicated that the MtuB2011probe advantageously was highly specific for Actinomycetes organisms.

The following Example describes additional methods used to demonstratespecificity of the probe. More particularly, the following proceduresshowed that the MtuB2011 probe did not cross hybridize with lysates fromnon-phylogentically related organisms.

EXAMPLE 3 Absence of Cross Hvbridization with Phylogentically UnrelatedOrganisms

Hybridization assays were conducted using the AE-labeled probes andhelper oligonucleotides according to the procedures described in theprevious Example except that lysates containing rRNA isolated fromnumerous diverse species served as target nucleic acids. Results of theprocedure are presented in Table 3. A pan-fungal probe having thesequence GTCTGGACCTGGTGAGTTTCCC (SEQ ID NO:7), and methoxy helperoligonucleotides having the sequences CGUGUUGAGUCAAAUUAAGCCGC (SEQ IDNO:8) and GCUCUCAAUCUGUCAAUCCUUAUUGU (SEQ ID NO:9) were used as positivecontrols to detect fungal rRNAs. Results obtained in hybridizationprocedures that employed rRNA targets from fungal organisms arepresented in Table 4.

TABLE 3 Hybridization of the MtuB2011 Probe with rRNA from a Collectionof Phylogenetically Non-Related Organisms Hybridization Results rRNASource Pan-Bacterial Actinomycetes Fractional *GP# (Organism) Probe(RLU) Probe (RLU) Hybridization (%) #234 Acinetobacter calcoaceticus4215 629 15 #233 Acinetobacter lwoffi 4345 551 13 #13 Bacillus brevis9293 818 9 #11 Bacillus subtilis 5184 860 17 #212 Bacteriodes fragilis7837 771 10 #226 Bacteroides ovatus 4107 563 14 #225 Bacteroidesthetaiotamicron 33410 553 2 #152 Citrobacter diversus 6835 744 11 #150Citrobacter freundii 9188 704 8 #192 Clostridium perfringens 7816 956 12#156 Enterobact gergoviae 3175 598 19 #153 Enterobacter aerogenes 8306733 9 #154 Enterobacter agglomerans 8753 752 9 #155 Enterobacter cloacae8150 732 9 #215 Enterobacter fragilis 7139 724 10 #46 Enterococcus avium9813 858 9 #27 Enterococcus casseliflavus 10339 667 6 #7 Enterococcuscecorum 5911 901 15 #15 Enterococcus dispar 91760 772 8 #85 Enterococcusdurans 8839 877 10 #82 Enterococcus faecalis 7910 879 11 #79Enterococcus faecium 7696 926 12 #23 Enterococcus faecium V1 5607 713 13#17 Enterococcus faecium V6 5761 755 13 #89 Enterococcus gallinarum 8682777 9 #81 Enterococcus hirae 6138 793 13 #45 Enterococcus malodoratus15407 737 5 #25 Enterococcus mundtii 9482 817 9 #26 Enterococcuspseudoavium 10042 703 7 #33 Enterococcus raffinosus 6662 675 10 #47Enterococcus sacchrolyticus 9017 844 9 #159 Escherichia coli 5587 598 11#161 Escherichiafergusonii 4598 585 13 #162 Escherichia hermanii 3267601 18 #217 Haemophilus influenzae 4157 635 15 #219 Haemophilusinfluenzae A 4182 596 14 #220 Haemphilus influenzae B 4247 570 13 #222Haemophilus parainfluenzae 25100 557 2 #188 Hafnia alvei 47770 941 2#163 Klebsiella oxytoca 3088 560 18 #164 Klebsiella ozaenae 5252 555 11#176 Klebsiella pbeumoniae 4631 4441 10 #178 Klebsiella rhinoscleromatis3567 474 13 #36 Lactobacillus acidophilus 20730 704 3 #56 Lactobacillusjensenii 6541 783 12 #9 Lactococcus lactis 36850 1022 3 #41 Listeriagrayi 2856 834 28 #72 Listeria ivanovii 6355 908 14 #31 Listeriamonocytogenes 1/2b 10102 677 7 #28 Listeria monocytogenes 4b 8225 685 8#73 Listeria seeligeri 7001 849 12 #40 Listeria welshimeri 5288 862 16#184 Morganella morganii 3852 440 11 #196 Neisseria gonorrhoea 6824 87213 #198 Neisseria meningitidis 20430 766 4 #191 Peptostreptococcus 6985824 12 anaerobius #179 Proteus mirabilis 5115 424 8 #183 Proteus penneri4285 927 22 #181 Proteus vulgaris 4883 433 9 #186 Providenciaalcalifaciens 44220 947 2 #187 Providencia rettgeri 31350 923 3 #185Providencia stuartii 13926 437 3 #200 Pseudomonas aeruginosa 5771 832 14#203 Pseudomonas cepacia 25590 982 4 #205 Pseudomonas fluorescens 8676773 9 #206 Pseudomonas maltophilia 7703 759 10 #209 Pseudomonasmendocina 17980 816 5 #208 Pseudomonas picketti 7954 991 12 #210Pseudomonas putida A 8216 788 10 #211 Pseudomonas stutzeri 7772 786 10#189 Salmonella enteritidis 20900 900 4 #216 Salmonella paratyphi 4253560 13 #165 Salmonella typhi 7967 1019 13 #166 Salmonella typhimurium5565 507 9 #170 Serratia liquifaciens 5801 551 9 #171 Serratiamarcescens 5093 471 9 #168 Shigella dysenterae 5233 764 15 #169 Shigellasonnei 5121 581 11 #49 Staphylococcus aureus 5964 838 14 #63Staphylococcus cohnii 6253 1066 17 #6 Staphylococcus delphini 6595 85713 #50 Staphylococcus epidermidis 8432 806 10 #62 Staphylococcushaemolyticus 5354 985 18 #61 Staphylococcus hominis 5593 1011 18 #69Staphylococcus hyicus 6193 877 14 #60 Staphylococcus intermedius 7501791 11 #59 Staphylococcus saprophyticus 4162 774 19 #39 Staphylococcusstimulants 6568 926 14 #67 Staphylococcus warner 4274 967 23 #53Streptococcus agalactiae 7295 845 12 #32 Streptococcus agalactiae la10144 691 7 #43 Streptococcus anginosis 8861 891 10 #16 Streptococcusavium 6366 687 11 #34 Streptococcus bovis 9298 664 7 #54 Streptococcusdysgalactiae 7095 744 10 #51 Streptococcus equi 12933 904 7 #80Streptococcus equinus 5259 888 17 #37 Streptococcus equisimilis 8123 77410 #97 Streptococcus grp C 9261 796 9 #98 Streptococcus grp G 7478 77010 #44 Streptococcus mutans 6874 939 14 #42 Streptococcus pneumoniae6153 869 14 #92 Streptococcus pyogenes 6723 886 13 #38 Streptococcussalivarius 8044 977 12 #35 Streptococcus sanguis 11918 705 6 #66Streptococcus sp gp F2 6921 894 13 #3 Streptococcus sp. Gp. B, II 154915 15 #5 Streptococcus uberis 6067 879 14 #173 Yersinia enterocolitica6165 521 8 #175 Yersinia intermedia 5246 460 9 #174 Yersiniapseudotuberculosis 5564 418 8 *“GP#” entries indicate master log numbersfor Gen-Probe Incorporated.

TABLE 4 Hybridization of the MtuB2011 Probe with rRNA from a Collectionof Fungal Organisms rRNA Source Pan-Bacterial MtuB2011 Fungal ProbeHybrid GP#* (Organism) Probe (RLU) Probe (RLU) (RLU) (%) F-932Arachnoitus flavoluteus 481 417 232076 0.2 F-906 Arachnoitus flavus 364395 348387 0.1 F-899 Aspergillus fumigatus 382 456 419831 0.1 F-907Aspergillus niger 194 411 650747 0.1 F-930 Auxarthron thaxteri 301 399494055 0.1 F-1022 Blastomyces dermatitidis 296 405 422465 0.1 715Candida albicans 369 333 327951 0.1 1123 Candida glabrata 1419 139845039 3.1 717 Candida parapsillosis 352 374 312482 0.1 1091 Candidatropicalis 1566 1437 24023 6 F-1399 Coccidioides immitis 303 329 1419560.2 F-900 Cryptococcus neoformans 316 302 452943 0.1 F-965 Gymnoascusdugwayenis 317 317 506033 0.1 F-968 Histoplasma capsulatum 254 255346283 0.1 F-933 Myxotrichum deflexum 267 319 366688 0.1 F-934Oidiodendron echinulatum 238 262 322685 0.1 716 Candida krusei 669 43271371 0.6 1087 Candida pseudotropicalis 243 35 79868 0 384 Saccharomycescerevisiae 116 76 75954 0.1 1080 Candida guilliermondii 320 203 650110.3 “GP#” identifies organisms by master log numbers for Gen-ProbeIncorporated.

The results presented in Table 3 and Table 4 confirmed that the MtuB2011probe did not cross hybridize with rRNA from numerous unrelatedbacterial and fungal species. Taken together with the positivehybridization results presented in the Table 2, it was clear that theprobe was highly specific for rRNA of the Actinomycetes.

These results confirmed that the novel probes disclosed herein werecapable of detecting the Actinomycetes. Moreover, the probes werecapable of distinguishing Actinomycetes from organisms that werephylogenetically closely related.

This invention has been described with reference to a number of specificexamples and embodiments thereof. Of course, a number of differentembodiments of the present invention will suggest themselves to thosehaving ordinary skill in the art upon review of the foregoing detaileddescription. Thus, the true scope of the present invention is to bedetermined upon reference to the appended claims.

19 1 29 DNA Actinomycetes 1 cgagcatctt tactcgtagt gcaatttcg 29 2 25 RNAActinomycetes 2 ccgagucugu gguugagaca guggg 25 3 25 RNA Actinomycetes 3ggucuuuccg uccugccgcg cguaa 25 4 16 RNA Pan-bacterial 4 cgacaaggaauuucgc 16 5 17 RNA Pan-bacterial 5 uaccuuagga ccguuau 17 6 16 RNAPan-bacterial 6 caggucggaa cuuacc 16 7 22 DNA Pan-fungal 7 gtctggacctggtgagtttc cc 22 8 23 RNA Pan-fungal 8 cguguugagu caaauuaagc cgc 23 9 26RNA Pan-fungal 9 gcucucaauc ugucaauccu uauugu 26 10 79 DNA Actinomycetes10 ggtctttccg tcctgccgcg cgtaacgagc atctttactc gtagtgcaat ttcgccgagt 60ctgtggttga gacagtggg 79 11 79 RNA Actinomycetes 11 cccacugucu caaccacagacucggcgaaa uugcacuacg aguaaagaug cucguuacgc 60 gcggcaggac ggaaagacc 7912 86 RNA S. aureus 12 uugggcacug ucucaacgag agacucggug aaaucauaguaccugugaag augcagguua 60 cccgcgacag gacggaaaga ccccgu 86 13 86 RNA E.coli 13 ggccaggcug ucuccacccg agacucagug aaauugaacu cgcugugaagaugcagugua 60 cccgcggcaa gacggaaaga ccccgu 86 14 85 RNA Frankia sp 14uuucccacug ucucaaccac agacucggcg aaauugcauu acgaguaaag augcucguua 60cgcgcggcag gacggaaaga ccccg 85 15 83 RNA M. tuberculosis misc_feature(1)...(83) n=a, g, c, u 15 uucucaacug ucucaaccau agacucggcg aaauugcacuacgaguaaag augcucgnac 60 gcgcggcagg ncgaaaanac ccc 83 16 61 RNA C.xerosis misc_feature (1)...(61) n=a, g, c, u 16 cccggcgaaa uugcgauacgaguaaagaug cunnuacgcg cggcaggacg aaaagacccc 60 g 61 17 86 RNA S. griseus17 uucucgacug ucucaaccau aggcccggug aaauugcacu acgaguaaag augcucguuu 60cgcgcagcag gacggaaaga ccccgg 86 18 85 RNA M. avium misc_feature(1)...(85) n=a, g, c, u 18 uucccaacug ucucaaccau agacucggcg aaauugcacuacgaguaaag augcucgnua 60 cgcgcggcag gncgaaaaga ccccg 85 19 85 RNA N.asteroides 19 uucucugcug ucucaaccac agacucggcg aaauugcauu acgaguaaagaugcucguua 60 cgcgcggcag gacgaaaaga ccccg 85

What is claimed is:
 1. An oligonucleotide probe that specificallyhybridizes to an Actinomycetes nucleic acid target region correspondingto E. coli 235 rRNA nucleotide positions 1986-2064 under a highstringency hybridization condition to form a detectable probe:targetduplex, said oligonucleotide probe having a length of up to 100nucleotides and comprising at least 17 contiguous nucleotides containedwithin the sequence of SEQ ID NO:10 or the complement thereof.
 2. Theoligonucleotide probe of claim 1, wherein said probe comprises at least25 contiguous nucleotides contained within the sequence of SEQ ID NO:10.3. The oligonucleotide probe of claim 2, wherein said probe comprises atleast 29 contiguous nucleotides contained within the sequence of SEQ IDNO:10.
 4. The oligonucleotide probe of claim 1, wherein the highstringency hybridization condition is provided by 0.48 M sodiumphosphate buffer, 0.1% sodium dodecyl sulfate, 1 mM each of EDTA andEGTA.
 5. The oligonucleotide probe of claim 1, wherein the highstringency hybridization condition is provided by 0.6 M LiCl, 1% lithiumlauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA.6. The oligonucleotide probe of claim 1, wherein said oligonucleotideprobe comprises DNA.
 7. The oligonucleotide probe of claim 1, whereinsaid oligonucleotide probe comprises at least one nucleotide analog. 8.The oligonucleotide probe of claim 7, wherein said at least onenucleotide analog comprises a methoxy group at the 2′ position of aribose moiety.
 9. The oligonucleotide probe of claim 1, wherein saidoligonucleotide probe has a sequence selected from the group consistingof SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complementthereof, and SEQ ID NO:3 or the complement thereof.
 10. Theoligonucleotide of claim 9, wherein said sequence is given by SEQ IDNO:2 or SEQ ID NO:3, said oligonucleotide being a helperoligonucleotide.
 11. The oligonucleotide probe of claim 9, furthercomprising a detectable label.
 12. The oligonucleotide probe of claim11, wherein the detectable label is a chemiluminescent label or aradiolabel.
 13. The oligonucleotide probe of claim 9, wherein saidsequence is given by SEQ ID NO:1.
 14. The oligonucleotide probe of claim13, wherein said oligonucleotide probe further comprises a detectablelabel.
 15. The oligonucleotide probe of claim 14, wherein the detectablelabel is an acridinium ester.
 16. A probe composition for detectingnucleic acids of Actinomycetes bacteria, comprising: an oligonucleotideprobe that hybridizes under a high stringency condition to anActinomycetes nucleic acid target region corresponding to E. coli 23SrRNA nucleotide positions 1986 - 2064 to form a detectable target:probeduplex, wherein said oligonucleotide probe has a length of up to 100nucleotide bases and comprises at least 17 contiguous nucleotidescontained within the sequence of SEQ ID NO: 10 or the complementthereof, and wherein under said hybridization condition saidoligonucleotide probe specifically hybridizes to nucleic acids presentin Corynebacterium aquaticum, Corynebacterium jeikieum, Corynebacteriumxerosis, Micrococcus luteus, Propionibacterium acnes, Mycobacteriumchelonae, Mycobacterium terrae, Mycobacterium intracellulare,Mycobacterium simiae, Mycobacterium avium, Mycobacterium scrofulaceum,Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium smegatis,Mycobacteriumfortuitum, Mycobacterium gastri, Mycobacterium xenopi,Mycobacterium marinum and Mycobacterium phlei.
 17. The probe compositionof claim 1, wherein said oligonucleotide probe has a length of up to 100nucleotide bases and comprises at least 25 contiguous nucleotidescontained within the sequence of SEQ ID NO:10 or the complement thereof.18. The probe composition of claim 16, wherein the oligonucleotide probecomprises DNA.
 19. The probe composition of claim 16, wherein said highstringency condition is provided by 0.48 M sodium phosphate buffer, 0.1%sodium dodecyl sulfate, 1 mM each of EDTA and EGTA.
 20. The probecomposition of claim 16, wherein said high stringency condition isprovided by 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithiumsuccinate and 10 mM each of EDTA and EGTA.
 21. The probe composition ofclaim 16, wherein said oligonucleotide probe comprises the sequence ofSEQ ID NO:1 or the complement thereof.
 22. The probe composition ofclaim 16, wherein the length of said oligonucleotide probe is up to 60bases.
 23. The probe composition of claim 16, wherein saidoligonucleotide probe has the length and sequence of SEQ ID NO:1. 24.The probe composition of claim 16, wherein said oligonucleotide probefurther comprises a detectable label.
 25. The probe composition of claim22, wherein said oligonucleotide probe further comprises a detectablelabel.
 26. The probe composition of claim 23, wherein saidoligonucleotide probe further comprises a detectable label.
 27. Theprobe composition of any one of claims 24, 25 or 26 wherein thedetectable label is a chemiluminescent label or a radiolabel.
 28. Theprobe composition of claim 27, wherein the chemiluminescent label is anacridinium ester.
 29. The probe composition of claim 27, furthercomprising at least one helper oligonucleotide that facilitatesformation of the detectable probe:target duplex under said hybridizationconditions.
 30. The probe composition of claim 29, wherein said at leastone helper oligonucleotide includes at least one nucleotide analog. 31.The probe composition of claim 30, wherein said at least one nucleotideanalog comprises a ribose moiety having a methoxy group disposed at the2′ position.
 32. The probe composition of claim 29, wherein said atleast one helper oligonucleotide has a sequence selected from the groupconsisting of SEQ ID NO:2 and SEQ ID NO:3.
 33. A method for detectingthe presence of Actinomycetes in a test sample, comprising the steps of:(a) providing to said test sample a probe composition comprising anoligonucleotide probe that hybridizes under a high stringency conditionto an Actinomycetes nucleic acid target region corresponding to E. coli23S rRNA nucleotide positions 1986-2064 to form a detectabletarget:probe duplex, said oligonucleotide probe having a length of up to100 nucleotide bases and comprising at least 25 contiguous nucleotidescontained within the sequence of SEQ ID NO:10 or the complement thereof,and wherein under said hybridization condition said oligonucleotideprobe specifically hybridizes to nucleic acids present inCorynebacterium aquaticum, Corynebacterium jeikieum, Corynebacteriumxerosis, Micrococcus luteus, Propionibacterium acnes, Mycobacteriumchelonae, Mycobacterium terrae, Mycobacterium intracellulare,Mycobacterium simiae, Mycobacterium avium, Mycobacterium scroffulaceum,Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium smegatis,Mycobacteriumfortuitum, Mycobacterium gastri, Mycobacterium xenopi,Mycobacterium marinum and Mycobacterium phlei; (b) hybridizing underhigh stringency conditions any Actinomycetes nucleic acid that may bepresent in the test sample with said probe composition to form aprobe:target duplex; and (c) detecting said probe:target duplex as anindicator of the presence of Actinomycetes in the test sample.
 34. Themethod of claim 33, wherein said test sample may comprise bacteria, andwherein before step (a) there is a step for releasing nucleic acid fromany bacteria that may be present in said test sample.
 35. The method ofclaim 33, wherein said test sample is a lysate.
 36. The method of claim33, wherein said high stringency hybridization conditions are providedby 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, 1 mMeach of EDTA and EGTA.
 37. The method of claim 33, wherein said highstringency hybridization conditions are provided by 0.6 M LiCl, 1%lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTAand EGTA.
 38. The method of claim 33, wherein the oligonucleotide probehas the length and sequence of SEQ ID NO:
 1. 39. The method of claim 38,wherein the oligonucleotide probe comprises a detectable label.
 40. Themethod of claim 39, wherein the detectable label is an acridinium ester,and wherein the detecting step comprises performing luminometry todetect any of said probe:target duplex.
 41. The method of claim 39,wherein said probe composition further comprises at least one helperoligonucleotide that facilitates formation of the probe:target duplex.42. The method of claim 41, wherein said at least one helperoligonucleotide is selected from the group consisting of SEQ ID NO:2 andSEQ ID NO:3.
 43. A kit for detecting the presence of Actinomycetesnucleic acids in a test sample, comprising: (a) a probe compositioncomprising an oligonucleotide probe that hybridizes under a highstringency condition to an Actinomycetes nucleic acid target regioncorresponding to E. coli 23S rRNA nucleotide positions 1986-2064 to forma detectable target:probe duplex, said oligonucleotide probe having alength of up to 100 nucleotide bases and comprising at least 25contiguous nucleotides contained within the sequence of SEQ ID NO:10 orthe complement thereof, and wherein under said hybridization conditionsaid oligonucleotide probe specifically hybridizes to nucleic acidspresent in Corynebacterium aquaticum, Corynebacterium jeikieum,Corynebacterium xerosis, Micrococcus luteus, Propionibacterium acnes,Mycobacterium chelonae, Mycobacterium terrae, Mycobacteriumintracellulare, Mycobacterium simiae, Mycobacterium avium, Mycobacteriumscrofulaceum, Mycobacterium gordonae, Mycobacterium kansasii,Mycobacterium smegatis, Mycobacteriumfortuitum, Mycobacterium gastri,Mycobacterium xenopi, Mycobacterium marinum and Mycobacterium phlei; and(b) printed instructions specifying, in order of implementation, thesteps to be followed for detecting said Actinomycetes nucleic acid bydetecting a complex between the oligonucleotide probe and anActinomycetes nucleic acid target, wherein said probe composition andsaid printed instructions are in packaged combination.